NMDA receptors with restricted mobility of the ligand binding domain

描述（由申请人提供）：NMDA受体是在脑生理学中具有关键作用的膜结合神经递质受体。它们是阿尔茨海默病和中风、智力迟钝以及成瘾、慢性疼痛、精神分裂症和癫痫的主要药物靶点。NMDA受体是谷氨酸激活的兴奋性离子通道。当神经递质谷氨酸与细胞外配体结合结构域（LBD）结合时，它们的激活反应开始，并随着膜嵌入的阳离子选择性孔的打开而达到高潮。构成NMDA受体活化反应的分子事件仍然未知，尽管它们在控制受体功能中起着基本作用。单受体活性的动力学研究已经确定，在结合激动剂后，NMDA受体在几种闭孔和开孔构象之间以可测量的速率循环。对分离的LBD的结构研究表明，配体结合在两个移动的叶之间的缝隙深处，并提出了这样的假设，即配体诱导的LBD闭合是沿着NMDA受体活化途径所需的步骤之一。本申请提出的实验将直接和具体地测试这一假设，通过检查活动受限的LBD的NMDA受体的活性。将在LBD叶的尖端处引入成对的半胱氨酸残基，作为将这些半胱氨酸残基相对于彼此锁定在限定距离处的手段。将通过在存在和不存在氧化剂的情况下以及在用各种长度的交联剂处理后对它们的宏观和单通道行为进行动力学分析来检查工程化受体。结果将提供有关身份的分子运动与NMDA受体激活的基本信息，并将提供有价值的洞察力有关的药物作用在该受体的可能机制。这些知识将形成合理控制NMDA受体活性作为中风，成瘾和精神疾病治疗策略所必需的概念基础。公共卫生相关性：该项目将提供有关NMDA受体变得活跃的机制的基本信息。这些信息是目前缺乏的，需要为急性和慢性神经变性（中风，阿尔茨海默病），成瘾，慢性疼痛，认知障碍和精神疾病的新的治疗方法的合理设计。

DESCRIPTION (provided by applicant): NMDA receptors are membrane-bound neurotransmitter receptors with critical roles in brain physiology. They are principal drug targets for Alzheimer's disease and stroke, mental retardation, and also addiction, chronic pain, schizophrenia and epilepsy. NMDA receptors are glutamate-activated, excitatory ion-channels. Their activation reaction is initiated when the neurotransmitter glutamate binds to extracellular, ligand-binding domains (LBDs) and culminates with the opening of a membrane-embedded cation-selective pore. The molecular events that make up the activation reaction of NMDA receptors remain unknown, despite their fundamental role in controlling receptor function. Kinetic studies of single-receptor activity have established that after binding agonists, NMDA receptors cycle with measurable rates between several closed-pore and open-pore conformations. Structural studies of the isolated LBDs have shown that ligands bind deep inside a crevice between two mobile lobes and have put forth the hypothesis that ligand-induced closure of the LBD is one of the required steps along the NMDA receptor activation pathway. This application proposes experiments that will directly and specifically test this hypothesis by examining the activity of NMDA receptors with restricted mobility of the LBDs. Pairs of cysteine residues will be introduced at the tips of the LBDs lobes as a means to lock these at defined distances with respect to one another. The engineered receptors will be examined by kinetic analyses of their macroscopic and single-channel behaviors in the presence and absence of oxidizing agents, and following treatment with crosslinking reagents of various lengths. The results will provide basic information regarding the identity of the molecular motions associated with NMDA receptor activation and will afford valuable insight regarding possible mechanisms of drug action at this receptor. This knowledge will form the conceptual foundation necessary to rationally control NMDA receptor activities as a therapeutic strategy for stroke, addiction, and mental illness. PUBLIC HEALTH RELEVANCE: This project will provide fundamental information regarding the mechanism by which NMDA receptors become active. This information is currently lacking and is needed for the rational design of novel therapeutic approaches for acute and chronic neurodegeneration (stroke, Alzheimer's disease), addiction, chronic pain, cognitive disorders and mental illness.